PROJECT SUMMARY Integrin ?IIb?3 (GP IIb-IIIa) is the platelet receptor for fibrinogen and is required for platelet aggregation during hemostasis. Fibrinogen binding to platelets is regulated by interactions of specific intracellular proteins, including talin and kindlin-3, with the ?3 cytoplasmic tail. In contrast, proteins that might interact with the ?IIb tail to regulate fibrinogen binding are relatively unexplored. We have found that human and mouse platelets and endothelial cells express the 40 kDa protein, SHARPIN. Studies with human platelets as well as with platelets and megakaryocytes derived from human induced pluripotent stem cells have revealed that SHARPIN can interact directly with either the ?IIb tail or with two other proteins to constitute the linear ubiquitination chain assembly complex (LUBAC). In fact, stimulation of platelets by traditional hemostatic agonists, such as thrombin, or by inflammatory agonists, such as lipopolysaccharide or soluble CD40 ligand (sCD40L), triggers both fibrinogen binding to ?IIb?3 and Met1-linked linear ubiquitination of IKK? (NEMO) to promote NF-kB pathway signaling. SHARPIN knockdown by shRNA in megakaryocytes and platelets results in decreased agonist-induced, linear ubiquitination of NEMO, but increased fibrinogen binding to ?IIb?3, MHC Class I expression, and release of endogenous sCD40L. Here we will test the hypothesis that SHARPIN?s mutually exclusive interactions with integrin ? tails or LUBAC regulate critical platelet and/or endothelial cell responses during hemostasis, thrombosis, inflammation and angiogenesis. Aim 1 will use advanced techniques, including optogenetics, to determine the stoichiometry of SHARPIN and ?IIb?3 in platelets and to test the functional effects of enforcing SHARPIN interactions with either ?IIb or LUBAC. Platelet-specific SHARPIN knockout mice will be generated in order to test the requirement for platelet SHARPIN in hemostasis, thrombosis and inflammation using a range of mouse models. Aim 2 will determine the role of SHARPIN in the adhesive and angiogenic functions of integrin ?V?3 and in NF-kB pathway signaling in endothelial cells. Endothelial cell SHARPIN will be specifically and conditionally knocked out in mice, and lung microvascular endothelial cells from these mice will be evaluated for ?V?3-dependent adhesive responses and for angiogenic sprouting. The effects of deleting endothelial cell SHARPIN in vivo will be determined using established mouse models of developmental and pathological angiogenesis. This project will make heavy use of the Hemostasis, Thrombosis, and Inflammation Models Core and it will collaborate with all other projects in this Program to achieve its aims. Altogether, these studies will provide a comprehensive test of the central hypothesis and establish new mechanistic insights into the regulation of integrin and immune signaling by SHARPIN in vascular cells, with clear implications for hemostasis, thrombosis, inflammation and angiogenesis.